Stress responses, outer membrane permeability control and antimicrobial resistance in Enterobacteriaceae 2 3

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Stress responses, outer membrane permeability control and antimicrobial resistance in Enterobacteriaceae 2 3

Sushovan Dam, Jean-Marie Pages, Muriel Masi

To cite this version:

Sushovan Dam, Jean-Marie Pages, Muriel Masi. Stress responses, outer membrane permeability control and antimicrobial resistance in Enterobacteriaceae 2 3. Microbiology, Microbiology Society, 2018,

�10.1099/mic.0.000613�. �hal-01840466�

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Stress responses, outer membrane permeability control and antimicrobial resistance in

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Enterobacteriaceae

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Sushovan Dam, Jean-Marie Pagès* and Muriel Masi

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UMR_MD-1, Aix-Marseille Univ. & IRBA, 27 Boulevard Jean Moulin, 13005 Marseille,

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France.

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*Corresponding author:

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jean-marie.pages@univ-amu.fr 12

+33 (0)4 91 32 46 97

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Key words: Enterobacteriaceae, envelope stress responses, outer membrane permeability,

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porins, drug translocation, multidrug resistance.

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Category: Regulation

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Word count (from introduction to conclusion):

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Abbreviations: outer membrane (OM), inner membrane (IMI), peptidoglycan (PG),

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lipopolysaccharide (LPS), antimicrobial resistance (AMR), multidrug resistance (MDR),

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envelope stress response (ESR), two-component system (TCS), small regulatory RNA

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(sRNA).

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Abstract

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Bacteria have evolved several strategies to survive a myriad of harmful conditions in the

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environment and in hosts. In Gram-negative bacteria, responses to nutrient limitation,

29

oxidative or nitrosative stress, envelope stress, exposure to antimicrobials and other growth-

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limiting stresses have been linked to the development of antimicrobial resistance. This results

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from the activation of protective changes to cell physiology (decreased outer membrane

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permeability), resistance transporters (drug efflux pumps), resistant lifestyles (biofilms,

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persistence) and/or resistance mutations (target mutations, production of antibiotic

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modification/degradation enzymes). In targeting and interfering with essential physiological

35

mechanisms, antimicrobials themselves are considered as stresses to which protective

36

responses have also evolved. In this review, we focus on envelope stress responses that affect

37

the expression of outer membrane porins and their impact on antimicrobial resistance. We

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also discuss evidences that indicate the role of antimicrobials as signaling molecules in

39

activating envelope stress responses.

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Introduction

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Antimicrobial resistance (AMR) is broadly recognized as a growing threat for human health

42

[1, 2, 3]. As such, increasing antibiotic treatment failures due to multidrug resistant (MDR)

43

bacteria have stirred the urgent need to better understand the underlying molecular

44

mechanisms and promote innovation with the development of new antibiotics and alternative

45

therapies [4, 5]. The efficacy of antibacterial compounds depends on their capacity to reach

46

inhibitory concentrations at the vicinity of their target. This is particularly challenging for

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drugs directed against Gram-negative bacteria, which exhibit a complex envelope comprising

48

two membranes and transmembrane efflux pumps [6, 7]. The Gram-negative envelope

49

comprises an inner membrane (IM), which is a symmetric phospholipid bilayer; a thin

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peptidoglycan (PG) layer ensuring the cell shape; and an outer membrane (OM) that is an

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asymmetric bilayer, composed of an inner leaflet of phospholipids and an outer leaflet of

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lipopolysaccharide (LPS) [8]. The OM is a barrier to both hydrophobic and hydrophilic

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compounds, including necessary nutrients, metabolic substrates and antimicrobials, but

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access is provided by the water filled

β-barrel channels called porins [6, 9, 10, 11, 12]. In 55

Escherichia coli, the channels of the general porins OmpF and OmpC are size restricted and

56

show a preference for passage of hydrophilic charged compounds, including antibiotics such

57

as

β-lactams and fluoroquinolones. These porins are conserved throughout the phylum of γ

-

58

proteobacteria [13]. Additionally, tripartite RND (Resistance-Nodulation-cell Division)

59

efflux pumps, such as AcrAB-TolC in E. coli, play a major role in removing antibiotics from

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the periplasm [7, 12]. Not surprisingly, MDR clinical isolates of Enterobacteriaceae

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generally exhibit porin loss and/or increased efflux, which act in synergy to reduce the

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intracellular accumulation of antibiotics below the threshold that would be efficient for

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activity [10].

64

Given the importance of the OM in controlling the uptake of beneficial as well as toxic

65

compounds, one can expect that the expression of porins depends on environmental stresses

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and is well-coordinated at the transcriptional and post-transcriptional levels [10, 14-17]. In

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this review, we will address the porin-mediated influx of antibiotics and give a perspective on

68

the factors, including major regulatory pathways and antibiotic stresses, which control porin

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expression in E. coli and closely relative Enterobacteriaceae. Additionally, we will discuss

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the recent clinical data that illustrate the bacterial strategies using porins modifications to

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limit antibiotic entry.

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Antibiotic stresses

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Bacteria are present in a wide range of environments in which they are exposed to diverse

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toxic compounds or growth-limiting conditions. These include antibiotics used in the medical

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environment and agricultural settings. The last few decades have been marked by the constant

77

increase of (multi)drug resistant clinical isolates to which we responded by increasing

78

antibiotic dosing. Therefore, antibiotics are present almost everywhere at different

79

concentrations [18]. Although MDR still emerges from bacterial exposure to antibiotic

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concentrations that are higher than the minimal inhibitory concentrations (MIC, defined as

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the lowest concentration of a drug that inhibit bacterial growth in defined laboratory

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conditions), the effects of subinhibitory concentrations on bacterial physiology and AMR was

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mostly disregarded. Importantly, studies in this field have shown that low antibiotic

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concentrations affect bacteria at least at four different levels: as selectors of resistance (by

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enriching resistant bacteria within populations and selecting for de novo resistance mutations)

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[19]; (ii) as contributors of genetic and phenotypic heterogeneity [20]; (iii) as intercellular

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signals [21]; (iv) as inducers of persistence [22]. In this regard, Viveiros and colleagues have

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demonstrated the induction of high-level resistance to tetracycline (TET) in susceptible E.

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coli K12 obtained by gradual, step-wise increase exposure to subinhibitory concentrations of

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the antibiotic [23]. Increased expression of the AcrAB efflux pump was found responsible for

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resistance to TET, which could also be reversed by the use of the efflux pump inhibitor

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phenylalanine-arginine-β-naphthylamide (PAβN). Interestingly, the TET-resistant strain also

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exhibited MDR due to repression of OmpF and OmpC expression [24]. Important questions

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arise from this and other related studies. First is whether the target for signaling resistance is

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the same as the target that is inhibited by the antibiotic. In case the antibiotic itself but not a

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secondary metabolite is the signaling molecule, this could be determined by examining

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whether the response is alleviated by a target mutation that prevents drug binding. Second is

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whether and how the antibiotic (or a secondary metabolite) interferes with the ESRs

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described above. Here, comparative transcriptomics between susceptible and resistant strains

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would be a valuable tool to answer this question.

101 102

Global regulators

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In Enterobacteriaceae, the development of MDR is under positive regulation by global

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transcriptional activators that include members of Ara/XylS superfamily such as MarA,

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RamA (absent in E. coli) and Rob as well as the oxidative stress regulon SoxSR [10, 25-29].

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Mutations in the corresponding genes are well-documented and induce the overproduction of

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efflux pumps with concomitant repression of porin expression both directly and indirectly

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[10]. These mechanisms are reviewed in details in Davin et al. [10]. Negative regulation by

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repressors of porins also plays a major role. OmpX is a small OM channel [30], of which

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overexpression is associated with a decreased expression of Omp36 (the OmpC ortholog of

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Enterobacter aerogenes) and a decreased susceptibility to β-lactams [31, 32]. Studies have

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indicated that expression OmpX itself is controlled by a number of environmental factors

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including salicylate via MarA and paraquat via SoxS [33] A very rapid MarA-dependent

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response pathway for upregulation of ompX has been shown to occur within 60-120 min upon

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cell exposure to salicylate [32]. This work by Dupont et al. identified dramatic decrease in

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OmpF levels, as a first line of defense, with simultaneous development of resistance to

β- 117

lactams and fluoroquinolones by altering OM permeability.

118 119

Envelope stress responses

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All living organisms have stress responses that allow them to sense and respond to

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environmental damaging conditions by remodeling gene expression. As such, Gram-negative

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bacteria possess stress responses that are uniquely targeted to the cell envelope, including

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membranes and cell wall. These envelope stress responses (ESRs) are the EnvZ/OmpR,

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CpxAR (Cpx), BaeRS, and Rcs phosphorelays, the stress responsive alternative sigma factor

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σ^E

, and the phage shock response [34-37] in E. coli and closely related Enterobacteriaceae.

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Each of these ESRs is activated following the perturbation of particular components of the

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envelope or exposure to particular environmental stresses. Although ESRs are important for

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reacting to damaging conditions, stress proteins also play important roles in the maintenance

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of basic cellular physiology [38, 39]. This is particularly true for the

σ^E

-dependent stress

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response in E. coli, as the rpoE gene, which encodes σ

^E

, is essential for viability [40]. Here,

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we will essentially focus on ESRs that impact on AMR by regulating porin expression

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together with many other targets (regulons) — namely EnvZ/OmpR, Cpx and σ

^E

(see below

133

and key figure). Additionally, with the recent highlights and advances in RNA-based

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techniques [41], the repertoire of small regulatory RNAs (sRNAs) has vastly increased so as

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to and their impact on the OM is continuously emerging [15, 17]. sRNAs alter gene

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expression, allowing fast adjustment to different growth conditions [42]. Noteworthy, ESRs

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are often interconnected, regulate and are regulated by sRNAs in order control target genes

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both at the transcriptional and post-transcriptional levels [15-17, 43, 44] (see below and key

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figure).

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Osmolarity was one of the earliest stresses described to influence OmpF and OmpC

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expression via the EnvZ/OmpR two-component system (TCS) [45, 46]. EnvZ is a membrane-

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bound sensor kinase, and OmpR is a cytosolic response regulator, which binds to the

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promoter region of the porin genes. Upon activation, EnvZ autophosphorylates and the high

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energy phosphoryl group from EnvZ is subsequently transferred to a conserved Asp residue

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on OmpR. Phosphorylated OmpR (OmpR~P) serves as a transcription factor that

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differentially modulates the expression of the ompF and ompC porin genes [45]. At low

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osmolarity, high levels of OmpR~P activates ompF transcription, whereas at high osmolarity,

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low levels of OmpR~P represses ompF transcription and activates ompC transcription [47].

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This differential production of OmpF and OmpC is consistent with that in high osmolarity

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environments, such as in the hosts where nutrients are abundant the least permeable pore

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channel OmpC is predominant, thus limiting the uptake of toxic bile salts; whereas in low

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osmolarity environments where nutrients are scarce, the most permeable pore channel OmpF

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is expressed [6]. OmpF and OmpC transcriptional regulation by EnvZ/OmpR is also triggered

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by local anesthetics, pH, and nutrient limitation [46].

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Accumulation of misfolded OM proteins in the periplasm, presumably reflecting problems in

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protein assembly or transport across the IM, can be detected by regulatory sensors that

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activate either the Cpx TCS or the alternative sigma factor

σ^E

.

σ^E

and Cpx are the two major

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regulation pathways that control the envelop integrity with overlapping regulon members

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[48-51] but respond to different inducing cues [35]. It is possible that these poorly defined

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signals (see below) act by causing accumulation of misfolded proteins. However, misfolded

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proteins are not the inducing signal per se, as some induce

σ^E

but not Cpx and vice versa.

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Recent studies rather suggest that Cpx responds to IM perturbations, while σ

^E

is activated by

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signals at the OM. The Cpx system comprises the CpxA sensor kinase and response regulator

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CpxR. Envelope stresses including alkaline pH, periplasmic protein misfolding, IM

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abnormalities such as misfolded transporters or accumulation of the lipid II precursor, induce

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the dissociation of the accessory protein CpxP from CpxA, trigger CpxA-mediated

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phosphorylation of CpxR, and altered expression of protein foldases and proteases,

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respiratory complexes, IM transporters, and cell wall biogenesis enzymes [37, 48, 49], all of

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which affect resistance to a number of antibiotics, particularly aminoglycosides and

β- 170

lactams [37, 49, 52-54]. The Cpx-mediated regulation of porins occurs at several levels. At

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the transcriptional level, CpxR~P has been shown to bind directly the ompF and ompC

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promoters [55]. More recently, it has been found that the small IM protein MzrA connects

173

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Cpx and EnvZ/OmpR [56]. In this pathway and upon the activation of Cpx, MzrA interacts

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directly with EnvZ, which in turn, stabilizes OmpR~P [57]. In sensing different signals, the

175

interconnection between Cpx and EnvZ/OmpR allows cells to adapt to diverse environmental

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stresses. Finally, although Cpx contributes to AMR by regulating a number of genes [37, 49,

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52-54], its precise role and that of other TCSs in the development of MDR in clinical isolates

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are still poorly documented [58]. On the other hand, the stress responsive sigma factor σ

^E

is

179

induced by stresses that disturb the OM and its regulon members comprise genes that

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facilitate the biogenesis of OM components, including proteins, lipoproteins and LPS [59-

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67]. In the absence of inducing signals,

σ^E

is held at the cytoplasmic side of the IM by the

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anti-sigma factor RseA. At the periplasmic side of the IM, RseB binds to RseA, thus

183

enhancing the inhibition of

σ^E

. Upon activation,

σ^E

is released from RseA by a proteolytic

184

cascade that starts with the sequential degradation of the periplasmic and transmembrane

185

domains of RseA by DegS and RseP, respectively, followed by the degradation of the

186

cytoplasmic domain of RseA by ClpXP [68]. Interestingly, proteolysis of RseA is triggered

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by the binding of a conserved peptide found at the C-terminus of OM proteins, which is

188

normally buried in folded porin trimers, to DegS in conjunction with the release of RseB

189

from RseA upon binding of LPS intermediates [69, 70]. Of note, the σ

^E

-dependent repression

190

of porin synthesis only occurs at the post-transcriptional level, wherein base-paring sRNAs

191

inhibits translation of omp mRNAs (see below) in order to maintain the envelope homeostasis

192

under stress conditions, as porins are major abundant proteins under normal growth

193

conditions [6].

194

The post-transcriptional repression of OmpF by the sRNA MicF has been discovered in 1984

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[71-73]. This 93-nucleotide (nt) RNA is transcribed in the opposite direction to the ompC

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gene and acts by direct base-pairing to a region that encompasses the ribosome binding site

197

(RBS) and the start codon of the ompF mRNA, thus preventing translation initiation [74].

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The expression of the MicF sRNA itself is subjected to multiple signals and regulatory

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pathways [75]. Positive regulation includes EnvZ/OmpR in high osmolarity conditions [76],

200

SoxS in response to oxidative stress [77] and MarA in response to antibiotic stress [25]. The

201

109-nt MicC sRNA has been identified more recently and shown to repress OmpC by direct

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base-pairing to a 5’ untranslated region of the ompC mRNA [78]. Interestingly, micC is

203

transcribed in the opposite direction to the ompN gene that encodes a quiescent porin

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homologous to OmpF and OmpC [79]. We have recently shown that ompN and micC are

205

subjected to dual regulation upon exposure to certain antimicrobials such as

β-lactams in a 206

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σ^E

-dependent manner [80]. This is consistent with that ompN-micC and ompC-micF share

207

similar genetic organization and that ompC and micF are co-induced under specific

208

conditions (i. e. high osmolarity via EnvZ/OmpR). The last decade has been marked by the

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identification and characterization of several sRNAs. These are differentially expressed and

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have been assigned to various important regulatory pathways in E. coli and Salmonella.

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Interestingly, many pathways regulate and are regulated by sRNAs [43, 44]. A prime

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example is EnvZ/OmpR, which activates the expression of MicF (that target ompF), OmrA

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and OmrB (that target ompT and mRNA of OM channels for iron-siderophore complexes)

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[81]; OmrA and OmrB, in turn, repress the translation of the ompR mRNA, creating a

215

negative feedback loop [82]. Others examples include the well-conserved

σ^E

-regulated

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sRNAs RybB (that target ompC and lamB in E. coli; ompN and ompW in Salmonella), MicA

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(ompA), RseX (ompC and ompA), CyaR (ompX) and MicL (that represses translation of the

218

major OM lipoprotein Lpp) [43, 66, 83-90] (key figure). Of note all these sRNAs are trans-

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acting, function by imperfect base pairing with multiple mRNA targets and require the help

220

of the RNA chaperone Hfq [15-17].

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Porin alterations in clinical isolates

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Combined regulations contributed by different stressors leads to hampering of the drug

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accumulation inside cells under the threshold for bacterial death. In one such study in K.

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pneumoniae, preferential expression of OmpK37 was detected in porin-deficient strains [92].

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Amino acid sequencing showed that OmpK37 is highly homologous to quiescent porins

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OmpS2 from Salmonella enterica serovar Typhimurium and OmpN from E. coli. Liposome

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swelling assay with purified porins determined that OmpK37 also has a narrower pore, which

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was responsible for higher MICs of cefotaxime and cefoxitin antibiotics because of lower

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drug diffusion. A very recent study identified mutation in the pho regulon of an extensively

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drug resistant strain of K. pneumoniae demonstrating downregulation of phoE gene by

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mutations in phoR and phoB. Here the PhoE porin, which is normally involved in phosphate

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transport, promotes restoration of cefoxitin and carbapenem resistance [93]. This is an

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interesting example of a regulatory mutation that effects porin expression, and clinically

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favors AMR under antibiotic therapy.

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A wide array of chemicals including disinfectants and antibiotics has been shown to modulate

237

the OM permeability including expression of porins [94]. In addition, several studies have

238

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described the effect of imipenem on porin loss or loss of function mutations in clinical

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isolates of Enterobacteriaceae [58, 95-100].

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Porins are trimers of 16-stranded

β-barrels, each monomer formed of a central channel 241

constricted by loop 3 that folds inward, thereby restricting the size of the channel. The

242

presence of acidic residues in loop 3 facing a cluster of basic residues on the opposite side of

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the pore creates a strong transversal electric field [6, 101, 102]. This so-called eyelet or

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constriction region determines the channel size and ion selectivity, with OmpF being more

245

permeable than OmpC. This latter observation was first attributed to the OmpC pore being

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slightly more constricted in this porin compared to OmpF [101, 102]. Although the two

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porins share high sequence similarity, the pore interior is more negative in OmpC than in

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OmpF [102]. This can also account for the low permeability of OmpC for anionic β-lactams

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[103, 104]. Moreover, the replacement of all ten titratable residues that differ between OmpC

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and OmpF in the pore-lining region leads to the exchange of antibiotic permeation properties

251

[105]. Together, these structural and functional data clearly demonstrate that the charge

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distribution at pore linings, but not pore size, is a critical parameter that physiologically

253

distinguishes OmpC from OmpF.

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Functional mutations in porin genes leading to reduced permeability are another strategy

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found in MDR bacteria. In two documented cases,

β-lactam-resistant clinical isolates of

E.

256

aerogenes contained Omp36 (an OmpC homologue) that carried the mutation G112D in L3

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[106, 97]. The homologous mutation G119D in OmpF of E. coli narrows the size of the

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channel as the large side chain of Asp protrudes into the channel lumen and confers a drastic

259

reduction in

β-lactam susceptibility [107]. Consistently, the Omp36 G112D mutant of

E.

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aerogenes was characterized by a 3-fold decrease in ion conductance and a significant

261

decrease in cephalosporin sensitivity (e. g. MICs of cefotaxime, cefpirome, cefepime and

262

ceftazidime were 7 to 9 fold higher in the clinical isolate as compared to that in a sensitive

263

reference strain) and a cross resistance to carbapenems [106, 97]. Recent studies also found a

264

series of OmpC mutants that were isolated from a patient with chronic E. coli infection and

265

additive mutations that conferred increased resistance to a variety of antibiotics, including

266

cefotaxime, ceftazidime, imipenem, meropenem and ciprofloxacin [108, 109].

Low et al.

267

demonstrated that subtle changes in OmpC in clinical isolates of E. coli altered antibiotic

268

permeability and thus cell viability [108]. Seven isolates collected over a two year clinical

269

treatment exhibited increased levels of antibiotic resistance. These isolates exhibited the same

270

two mutations (D18E and S274F) in the OmpC porin with increased levels of antibiotic

271

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resistance, thus pointing towards the possible functional role of these mutations in antibiotic

272

influx.

273

It is worthwhile to note that from our knowledge, porin mutations causing reduced

274

permeability have only been described in OmpC-type porins in E coli and E aerogenes.

275

Interestingly, this type of porin is expressed under high osmolarity, the same environment the

276

bacteria encounters the hosts. This gives an essential outlook on the host induced

277

modifications that possibly occurs in these pathogens during infection. Heeding to this sort of

278

information can be highly beneficial for designing drugs with an improved diffusion across

279

the bacterial outer membrane.

280 281

Conclusion

282

It is noteworthy that the sRNA-mediated stress response mechanism has multiple benefits for

283

bacteria as compared to regulation by protein. Since sRNAs are produced during

284

transcription, the later stages of translation and post translational modification processes in

285

the cell is completely skipped proving to be time and energy efficient for the cell. Not to

286

forget the energy saved in porin assembly and discarding of misfolded proteins, which in

287

itself can induce another stress response mechanism.

288

Decreased porin expression has been observed as a rapid response to toxic molecules and

289

antibiotics within less than 60 minutes. Many sRNAs act at the post-transcriptional level,

290

which ensures a rapid response to stressful conditions. In addition, the versatility of sRNAs

291

ensures another level of gene regulation along with protein transcriptional regulators, thus

292

contributing to an additional layer of tighter regulation. Taking into account the major role of

293

the CpxAR and EnvZ/OmpR regulators in response to stressors such as antibiotics, it will be

294

interesting to develop some assays allowing the detection of these kinds of mutations inside

295

clinical isolate. This original diagnostic maybe used for determining the prevalence of these

296

regulation events in clinical strain that have undergone antibiotic stress.

297

Targeting the early transcriptional step of antibiotic stress response regulatory mechanism is

298

much more logical, especially when we have reports of OMP expression being regulated

299

(both up and downregulation) within 60 minutes of stress appearance [32]. This will

300

especially promote bypassing of aforementioned mutations in porins in clinical strains that

301

are selected during antibiotic treatment. Targeting of sRNA or sRNA regulators such as

302

MicF or Hfq can rejuvenate failing antimicrobial therapies in regards with membrane

303

impermeability. They can be original targets for increasing the efficiency of existing drugs by

304

providing fitness reduction in bacteria. As of now, a cyclic peptide RI20 has been identified

305

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to inhibit Hfq-mediated repression of gene, by binding with proximal binding site of Hfq

306

[110]. Another approach will be to inhibit sRNA interfering with porin expression that is

307

involved in drug translocation. Recently, a small molecule was used to target human

308

microRNA (miR)-525 precursors as an anti-cancer strategy [111]. This promising discovery

309

can be repeated in bacteria for manipulating sRNA levels, which may save the failing

310

antibiotic therapies.

311

Predictability of an efficient drug based on the SICAR (Structure Intracellular Concentration

312

Activity Relationship) concept, is a step up to efficient drug designing. Briefly, SICAR

313

connects the physicochemical drug properties to the efficacy of translocation through the

314

bacterial membrane and the resulting intracellular accumulation. To achieve this goal, an

315

extensive knowledge of the OM permeability control, including the contribution of sRNAs, is

316

required.

317

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Funding information: The research leading to the discussions presented here was conducted

318

as part of the Marie Curie Initial Training Network TRANSLOCATION consortium and has

319

received support from the ITN-2013-607694-Translocation (SD). This work was also

320

supported by Aix-Marseille Univ. and Service de Santé des Armées.

321 322

Acknowledgments: We thank all the members of the UMR_MD1, especially Estelle Dumont

323

and Julia Vergalli, for helpful discussions throughout this work.

324 325

Conflicts of interest: The authors declare no conflict of interest. The founding sponsors had

326

no role in the design of the study; in the collection, analyses, or interpretation of data; in the

327

writing of the manuscript, and in the decision to publish the results.

328

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Key figure: Major regulatory pathways of porin regulation in E. coli: EnvZ/OmpR [46],

608

CpxAR and sigma E (σ

^E

) [35] stress response systems are shown, along with known inducing

609

cues and targets relevant to porin regulation. The upregulation is shown with thick green

610

arrows, while the downregulation is shown with red lines. In the EnvZ/OmpR TCS,

611

activation of the response regulator OmpR results in phosphorylated and OmpR∼P

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downregulates the expression of OmpF both at the transcriptional and post-transcriptional

613

levels, the latter via the MicF sRNA. The mar-sox-rob regulons also downregulate OmpF

614

expression via MicF. Both the CpxAR and

σ^E

responses are activated by a variety of

615

envelope stresses. For clarity, only periplasmic misfolded OMPs are represented here. On one

616

hand, CpxR

∼

P alters expression of multiple genes, including that of micF. On the other hand,

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the anti-sigma factor RseA is degraded by the successive action of two proteases, DegS and

618

RseP at the periplasmic and the cytoplasmic site. Another protease, ClpXP specifically

619

degrades the cytoplasmic RseA portion bound to

σ^E

, leading to its release. A number of σ

^E

-

620

regulated sRNAs are indicated: MicC [78] downregulates OmpC and is coupled with ompN

621

upregulation [80]; sRNA regulation of porins via CyaR [90], IpeX [91], RseX [86] and RybB

622

[84, 88] are shown accompanied by their activators and porin targets; CyaR negatively

623

regulates the expression of single channeled porin OmpX [30], which in turn negatively

624

regulates the major porin OmpC. The details of all these interconnected pathways are

625

discussed thoroughly in the text.

626 627

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628